Glycosylphosphatidylinositol (GPI) serves as a novel membrane anchor for many eukaryotic proteins. Our laboratory has identified a series of GPI biosynthetic intermediates and proposed a biosynthetic pathway for the mammalian GPI anchor. These advances contributed to the elucidation of the molecular defect in Paroxysmal Nocturnal Hemoglobinuria (PNH), an acquired hematopoietic disorder affecting GPI anchor biosynthesis. Recently, our laboratory has cloned the human class H cDNA, one of the three genes (Class H, A, and C) required the initiation of GPI anchor biosynthesis, i.e. in the transfer of G1cNAc from UDP-G1cNAc to an inositol phospholipid (PI) acceptor. In the same year, the class A cDNA was independently cloned by another laboratory. These advances provide us with an excellent opportunity to study the first step of GPI anchor biosynthesis in molecular terms. Our aims are: 1) to characterize the class H protein. Antibodies against the class H protein will be produced. Western blot analysis, immunoprecipitation, and immunofluorescent microscopy will be used to define its subcellular localization, membrane association, membrane orientation, and association with other proteins; 2) to clone the mouse class H cDNA and the human class H gene. Sequence conservation between species and exon/intron structure will provide clues on the structural organization of the class H protein; 3) to characterize the class A protein. The potential association of the class H and A protein will be explored; 4) to elucidate the molecular defects in selected PNH patients. Since most PNH patients have class A defect, knowledge of missense or frameshift mutations in the class A cDNA will pinpoint important functional domains in the class A protein; 5) to clone the class C cDNA. In order to fully understand the transferase activity, the human class C cDNA will be identified by expression cloning; 6) to study the structure/function relationship of the UDP-G1cNAc:PI transferase. Once all three genes have been identified and their gene products expressed, photoaffinity labeling with azido-UDP-G1cNAc and a solid phase ELISA will be carried out to determine the specific function of these proteins. Finally, purified proteins will be used to reconstitute the transferase activity in vitro. These studies will be significantly enhance our understanding of the biochemistry, molecular biology, and cell biology of GPI anchor biosynthesis in normal and PNH cells.